PROJECT SUMMARY Our long-term goal is to define gene networks that enable neural stem cells to produce remarkably divergent cell types in CNS development. The combinatorial action of transcription factors is a prevalent strategy to achieve cellular complexity in CNS. However, the mechanisms underlying combinatorial action of transcription factors in controlling the expression of unique set of terminal differentiation genes for a specific cellular identity remain unclear in vertebrate CNS. In this proposal, we wish to tackle this important issue by focusing on the gene networks for the specification of spinal motor neurons, in which the developmental transcription codes are relatively well understood. LIM homeodomain proteins Lhx3 and Isl1 regulate motor neuron specification in combination by forming a hexameric complex, named MN-hexamer. The key hypothesis of this proposal is that MN-hexamer directly controls a battery of genes that control wide aspects of MN identity, including cholinergic neurotransmission, by coordinating the actions of retinoid signal and chromatin modifying enzymes during spinal cord development. We will test this hypothesis using an ensemble of molecular and biochemical methods, genetically engineered embryonic stem cells, chick embryos and mutant mice. Three specific aims are proposed to dissect the hypothesis; 1) To define the target genes of MN-hexamer that assign MN identity. 2) To investigate the regulation of cholinergic neuronal identity by similar hexameric complexes in spinal motor neurons and forebrain cholinergic neurons. 3) To define the role of RA in facilitating MN specification by MN-hexamer. Besides providing crucial insights into the generation of motor neurons and motor circuits, our studies will lay fundamental framework to study gene networks in creating the amazing cellular diversity during CNS development. These studies should also provide new tools for developing therapeutic strategies for the spinal cord injuries and diseases associated with impaired motor function, such as ALS (Lou Gehrig?s disease), and the cognitive disorders resulting from the loss of forebrain cholinergic neurons, such as Alzheimer?s diseases.